The invention provides nucleotide sequences which code for the opcA gene and a process for the fermentative preparation of amino acids, in particular L-lysine using coryneform bacteria in which the opcA gene is amplified.
Amino acids, in particular L-lysine, are used in human medicine and in the pharmaceuticals industry, but in particular in animal nutrition.
It is known that amino acids are prepared by fermentation from strains of coryneform bacteria, in particular Corynebacterium glutamicum. Because of their great importance, work is constantly being undertaken to improve the preparation processes. Improvements to the processes can relate to fermentation measures, such as e.g. stirring and supply of oxygen, or the composition of the nutrient media, such as e.g. the sugar concentration during the fermentation, or the working up to the product form by e.g. ion exchange chromatography, or the intrinsic output properties of the microorganism itself.
Methods of mutagenesis, selection and mutant selection are used to improve the output properties of these microorganisms. Strains which are resistant to antimetabolites, such as e.g. the lysine analogue S-(2-aminoethyl)-cysteine, or are auxotrophic for metabolites of regulatory importance and produce L-amino acids, such as e.g. L-lysine, are obtained in this manner. Methods of the recombinant DNA technique have also been employed for some years for improving the strain of Corynebacterium strains which produce amino acids.
The importance of the pentose phosphate cycle for the biosynthesis is known.
Thus Oishi and Aida (Agricultural and Biological Chemistry 29, 83-89 (1965)) already report on the xe2x80x9chexose monophosphate shuntxe2x80x9d of Brevibacterium ammoniagenes. Sugimoto and Shio (Agricultural and Biological Chemistry 51, 101-108 (1987)) report on the regulation of glucose 6-phosphate dehydrogenase in Brevibacterium flavum. Sugimoto and Shio (Agricultural and Biological Chemistry 51, 1257-11263 (1987)) report on the regulation of glucose 6-phosphate dehydrogenase in Brevibacterium flavum. 
JP-A-09224661 discloses the nucleotide sequence of the glucose 6-phosphate dehydrogenase gene, called zwf, of Brevibacterium flavum MJ-223 (FERM BP-1497). JP-A-09224661 describes the N-terminal amino acid sequence of the Zwf polypeptide as Met Val Ile Phe Gly Val Thr Gly Asp Leu Ala Arg Lys Lys Leu (SEQ ID NO: 24).
However, it has not been possible to confirm this.
Amino acids, in particular L-lysine, are used in human medicine, in the pharmaceuticals industry and in particular in animal nutrition. There is therefore a general interest in providing new improved processes for the preparation of amino acids, in particular L-lysine.
When L-lysine or lysine are mentioned in the following, not only the base but also the salts, such as e.g. lysine monohydrochloride or lysine sulfate, are also meant by this.
The invention provides an isolated polynucleotide from coryneform bacteria, comprising at least one polynucleotide sequence chosen from the group consisting of
a) polynucleotide which is identical to the extent of at least 70% to a polynucleotide which codes for polypeptides which comprise at least one of the amino acid sequences according to SEQ ID No. 3 or SEQ ID No. 5 or SEQ ID No. 8 or SEQ ID No. 10,
b) polynucleotide which codes for polypeptides which comprise amino acid sequences which are identical to the extent of at least 70% to the amino acid sequences according to SEQ ID No. 3 or SEQ ID No. 5 or according to SEQ ID No. 8 or SEQ ID No. 10,
c) polynucleotide which is complementary to the polynucleotides of a) or b), or
d) polynucleotide comprising at least 15 successive nucleotides of the polynucleotide sequences of a), b) or c).
The invention also provides the polynucleotide as described above, this polynucleotide preferably being a DNA which is capable of replication, comprising:
(i) one or more nucleotide sequence(s) chosen from the group consisting of SEQ ID No. 1, SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 9, or
(ii) at least one sequence which corresponds to sequence (i) within the range of the degeneration of the genetic code, or
(iii) at least one sequence which hybridizes with the sequence complementary to sequence (i) or (ii), and optionally
(iv) sense mutations of neutral function in (i).
The invention also provides
a polynucleotide comprising the nucleotide sequence as shown in SEQ ID No. 4 or SEQ ID No. 9,
a polynucleotide which codes for a polypeptide which comprises at least one of the amino acid sequences as shown in SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 8 or SEQ ID No. 10,
a vector containing the above polynucleotide,
and coryneform bacteria, serving as the host cell, which contain the vector.
The invention also provides polynucleotides which substantially comprise a polynucleotide sequence, which are obtainable by screening by means of hybridization of a corresponding gene library, which comprises the complete gene with the polynucleotide sequence corresponding to SEQ ID No. 4 or SEQ ID No. 9, with a probe which comprises the sequence of the polynucleotide mentioned, according to SEQ ID No. 4 or SEQ ID No. 9 or a fragment thereof, and isolation of the DNA sequence mentioned.
SEQ ID No. 1: DNA sequence isolated from Corynebacterium glutamicum ATCC13032.
SEQ ID No. 2: Amino acid sequence of the Zwf protein derived from SEQ ID No. 1.
SEQ ID No. 3: Amino acid sequence of the OpcA protein derived from SEQ ID No. 1.
SEQ ID No. 4: DNA sequence of the opcA gene of ATCC13032 taken from SEQ ID No. 1.
SEQ ID No. 5: Amino acid sequence of the OpcA protein derived from SEQ ID No. 4.
SEQ ID No. 6: DNA sequence isolated from Corynebacterium glutamicum ASO19.
SEQ ID No. 7: Amino acid sequence of the Zwf protein derived from SEQ ID No. 6.
SEQ ID No. 8: Amino acid sequence of the OpcA protein derived from SEQ ID No. 6.
SEQ ID No. 9: DNA sequence of the opcA gene of ASO19 taken from SEQ ID No. 6.
SEQ ID No. 10: Amino acid sequence of the OpcA protein derived from SEQ ID No. 9.
SEQ ID No. 11: Amino acid sequence of the N-terminus of the Zwf protein of the glucose 6-phosphate dehydrogenase from ATCC13032 which can be isolated.
SEQ ID No. 12: Amino acid sequence of the N-terminus of the OpcA protein of the glucose 6-phosphate dehydrogenase, which can be isolated from ATCC13032.